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[Preprint]. 2022 Nov 30:2022.11.26.518005. [Version 1] doi: 10.1101/2022.11.26.518005

FIGURE 2. Cloning-free generation and characterization of CPER-derived recombinant SARS-CoV-2.

FIGURE 2.

(A) Representative gel images of the overlapping cDNA fragments amplified from purified SARS-CoV-2 genomic RNAs of the indicated virus strains. The primer sets described by Torii et al. [18] conform to the genome sequences of the ancestral strain (WA1) and the selected Beta and Omicron variants of concern (VOCs) with 100% complementarity. MM, molecular marker.

(B) Schematic of the overlapping PCR strategy for site-directed mutagenesis in fragment 2 by using purified PCR product as a template (top panel), as well as representative gel images of the intermediate (2.1 and 2.2) and final (2mut) PCR products (bottom panel). MM, molecular marker.

(C) Sequencing confirmation of the integrity of the spike furin cleavage site of the passage 0 (P0) virus stocks from three independent virus rescues using the optimized CPER approach. aa, amino acids; nt, nucleotides.

(D) Plaque morphology on Vero E6-TMPRSS2 cells of recombinant Beta (rBeta) generated by optimized CPER as well as of its parental virus.

(E) Virus titers of the P0 stocks of CPER-derived recombinant WA1 (rWA1) and rBeta, collected at day 5 and day 4 post-transfection of the CPER product, respectively (n = 4).